Cellular toxicity associated with poly (ADP)-ribosylation reactions resulting from DNA damage contributes to pathogenesis of several types of diseases. Poly (ADP)-ribosylation reactions have been shown to be associated with the cellular damage occurring in neurodegenerative diseases, autoimmune diseases, infections and cancer.
Several mechanisms, including increases in nitric oxide may contribute to activation of poly (ADP-ribose) synthetase which catalyzes the poly (ADP)-ribosylation reaction. Cleaver J. E. & Morgan W. F., Mutat. Res. 257:1-18, 1991; Snyder S. H., Science, U.S.A., 265: 723, 1994; Zhang J. & Snyder S. H., Proc. Natl. Acad. Sci., U.S.A., 89:9382-9385, 1992; DeMurcia G., et al., Bio Essays, 13:455-462, 1991. Nitric oxide is produced in a variety of cell types including neurons and blood endothelial cells. Kandel E. R., Schwartz J. H, Jessell T. M., Principles of Neural Science. Third Edition, 1991, p191. Nitric oxide is also significant as a possible pathogenic agent for multiple populations of cells because, besides being toxic to the cells in which nitric oxide is produced, nitric oxide is also released into blood where it acts as a "local hormone". Id. In addition, nitric oxide is capable of readily passing across cell membranes into adjacent cells. Id.
Activation of poly (ADP-ribose) synthetase is reported to be associated with the pathogenesis of a variety of neurodegenerative disorders in response to the generation of toxic quantities of nitric oxide. Zhang J. et al., Science. U.S.A., 263:687-689, 1994. Generation of nitric oxide in neurons occurs in response to over-stimulation of NMDA receptors by naturally occurring excitotoxic agents present in the brain including, for example, glutamic and quinolinic acids. Inhibitors of nitric oxide production provide protection against the pathogenic effects of the excitatory agents. Dawson V. L., et al., Proc. Natl. Acad. Sci., U.S.A., 88:6368-6371, 1991.
Inhibition of poly (ADP-ribose) synthetase activity has also been associated with the action of anti-viral agents inhibiting gene expression in HIV-1, the virus causing AIDS. Yamagoe S., et al., Molec. Cell. Biol., 11:3522-3527, 1991. For example, both nicotinamide and benzamide, known inhibitors of poly (ADP-ribose) synthetase, when added to cultures of HIV-1 infected cells demonstrated significant antiviral activity. Id. Further support for such a mechanism mediating antiviral activity of nicotinamide comes from recent studies linking cellular HIV-1 infection with decreased levels of nicotinamide adenine dinucleotide (NAD) and a significant antiviral effect of nicotinamide treatment of HIV-infected cells in culture (Murray MF et al., Biochem. Biophys. Res. Commun., 210:954-959, 1995; and, Murray MF et al., Biochem. Biophys. Res. Commun., 212:126-131, 1995). In addition, relevant to HIV-1 associated encephalopathy, it has been reported that the HIV-1 coat protein, gP120, kills neurons in cell culture by a mechanism involving nitric oxide. Dawson V. L., et al., Proc. Natl. Acad. Sci.. U.S.A., 90:3256-3259, 1993. Like neuronal cells, toxicity of nitric oxide in HIV-1 infected cells, and other cells infected by virus of several types (Goldman N., et al., Cell 24:567-572, 1981; Dery C. V., et al., Virus Res. 4:313-329, 1986; Mansuri M. M. & Martin J. C., Ann. Rep. Med. Chem. 24:133-140, 1991) is at least partially caused by activating poly (ADP-ribose) synthetase.
Enhancement of poly-(ADP)-ribosylation has also been reported to be associated with the pathogenesis of certain forms of diabetes. Destruction of the beta cells of the pancreas, which cells make and release insulin, is also associated with nitric oxide toxicity resulting in overactivation of poly (ADP-ribose) synthetase. Kallman, B., et al., Life Sci. 51:671-678, 1992; Suarez-Pinzon W. L., et al., Endocrinology 134: 1006-1010, 1994.
Involvement of poly (ADP-ribose) synthetase in the development of certain cancers has also been reported. Borek C., et al., Proc. Natl. Acad. Sci.. U.S.A., 81:243-247, 1984; Tseng A. Jr., et al., Proc. Natl. Acad. Sci. U.S.A., 84:1107-1111, 1987; and Alderson T., Biolog. Revs. 65:623-641, 1990.
Despite the various reports establishing a pathogenic action of poly (ADP-ribose) synthetase induction, there are few, if any treatments which are suitable for administration to an individual and which result in therapeutic decreases in poly (ADP-ribose) synthetase. In preparations of crude or partially purified enzyme, nicotinamide and a number of benzamide derivatives are effective inhibitors of poly (ADP-ribose) synthetase. Banasik M., et al., J. Biol. Chem., 267:1569-1575, 1992. Despite their effectiveness at inhibiting poly (ADP-ribose) synthetase, nicotinamide and benzamide have only limited applicability for therapeutic applications because of their polar nature. Accordingly, millimolar concentrations of these compounds are required to achieve sufficient intracellular uptake and localization to appropriate intracellular target organelles such as the nucleus.
Nicotinamide is an intermediate in the metabolism of the amino acid tryptophan to nicotinamide adenine dinucleotide (NAD) and to nicotinuric acid. Tryptophan is metabolized by the kynurenine pathway, illustrated below, which branches to produce the excitotoxin quinolinic acid or nicotinamide which is subject to glycine conjugation to produce nicotinuric acid.
In the kynurenine pathway, tryptophan is converted by two sequential enzymatic reactions to kynurenine. Kynurenine is then converted either to kynurenic acid by the enzyme kynurenine transaminase, or to 3-hydroxykynurenine by the enzyme kynurenine hydroxylase. The enzyme kynureninase then converts 3-hydroxykynurenine to 3-hydroxyanthranilic acid, an intermediate in the production of the excitotoxin quinolinic acid. Indeed, substrate nonspecificity of the mammalian enzyme kynureninase (EC 3.7.1.3) is documented in the literature. Besides the two natural substrates already listed, other naturally occurring and synthetic substrates exist. For example, kynureninase also splits some .gamma.-oxy-aliphatic amino acids as well as some .gamma.-oxy-phenyl amino acids (Wiss O. and Fuchs H., Experientia, 12:472-473, 1950). It is this nonspecificity of the enzyme which may be used to therapeutic advantage by the design of drugs as potential substrates. ##STR1##
Nicotinylalanine (also referred to as .gamma.-(3-pyridyl-.gamma.-oxo-.alpha.-aminobutyrate; M. W.=194.1 Daltons) (Formula I)) is an inhibitor of both kynurenine hydroxylase and kynureninase. As a substrate for the enzyme kynureninase, nicotinylalanine is itself converted to nicotinamide. Decker R. H., et al., J. Biol. Chem., 238:1049-1053, 1963. ##STR2## Nicotinylalanine has an asymmetric carbon atom and accordingly exists in enantiomeric (2R and 2S) or racemic forms. Inhibition of the kynurenine hydroxylase and kynureninase enzymes not only reduces production of the excitotoxin quinolinic acid, but also increases the synthesis of kynurenic acid, a compound capable of inhibiting the effect of quinolinic acid. Decker, R. H., et al., J. Biol. Chem., 238:1049-1053, 1963; Moroni, F., et al., J. Neurochem., 57:1630-1635, 1991.
This enzyme-inhibitory activity of nicotinylalanine is discussed as the basis for the use of nicotinylalanine to protect against the neurotoxicity associated with metabolism of tryptophan and production of quinolinic acid in Pellicciari, R., et al., International application WO 91/17750.
Despite the use of nicotinylalanine to reduce toxicity associated with quinolinic acid, new methods and compositions are still needed to provide more effective reduction of cellular toxicity associated with poly (ADP) ribosylation reactions. Such methods and compositions are also needed to provide therapeutic increases in endogenous concentrations of nicotinamide which can effectively inhibit the activity of poly (ADP-ribose) synthetase reactions.
As a co-factor for the enzyme kynureninase, the active cellular form of B6 pyridoxal phosphate increases the rate of conversion of nicotinylalanine to nicotinamide (Takeuchi F. and Shibata Y., Biochem. J., 220: 693-699, 1984). Pyridoxal phosphate (PLP) deficiency is reported to occur in viral diseases, including HIV infection and its associated complications in AIDS (Baum M. K. et al., J. Acq. Imm. Def. Synd., 4:1122-1132, 1991), and in various cancers in mice (Gridley D. S. et al., J. Natl. Cancer Inst., 78:951-959, 1987; Ha C. et al., J. Nutr., 114:938-945, 1984) as well as in humans (Potera M. S. et al., Am. J. Clin. Nutr., 30:1677-1679, 1977). General descriptions of pathological conditions in humans underlying PLP deficiency have also been reported. (Serfontein W. J., U.S. Pat. No. 5,254,572, 1993).